The long term goal of this research is to better understand the mechanisms of intracellular pH (pHi) regulation in adult ventricular muscle. In this project the applicant will focus on two modulators of cardiac pHi control systems, vasoactive peptides (angiotensin II, endothelin 1) and chronic (4-5 days) acid-base disturbances (metabolic acidosis and alkalosis). These peptides are potent positive inotropic agents which exert a wide range of effects on the heart and are currently thought to exacerbate myocardial damage during ischemia/reperfusion. Their cardiac effects may be mediated, in part, by changes in pHi. Similarly, the effect of prolonged acidosis and alkalosis on cardiac pHi control systems may represent an important adaptive response to chronic myocardial ischemia. The applicants will test the hypothesis that both modulators alter the kinetic properties and pHi dependence of Na/H exchange, Na-HCO3 cotransport, and Na- independent Cl-HCO3 exchange. Experiments are also designed to identify the sarcolemmal receptor which mediates the pHi action of the peptides. The applicant will also investigate the intracellular signaling role of Cai, calmodulin, Ca/calmodulin-dependent protein kinase II, and protein kinase C in mediating the pHi effects of ATII and ET1. The experiments will be performed at 37oC on ventricular myocytes, enzymatically isolated from normal adult rabbits, guinea pigs, and humans (only rabbits will be used in the chronic acid-base studies). Intracellular activities of H+ and Na+ will be measured with the fluorescent indicators (SNARF-1, BCECF) and SBFI, respectively. In some experiments, measurement of pHi and Nai will be made during internal dialysis of cells with a suction pipette. A rapid solution switcher will be used for rapid (less than 3 msec) external application of the peptides.